Co-Production of Nattokinase and Poly ( -Glutamic Acid ... · industrial application in the fields...

Braz. Arch. Biol. Technol. v.58 n.5: pp. 718-724, Sept/Oct 2015

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Vol.58, n.5: pp. 718-724, September-October 2015 http://dx.doi.org/10.1590/S1516-89132015050172

ISSN 1516-8913 Printed in Brazil

BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY

A N I N T E R N A T I O N A L J O U R N A L

Co-Production of Nattokinase and Poly (γ-Glutamic Acid)

Under Solid-State Fermentation Using Soybean and Rice

Husk

Guangjun Nie, Zhubin Zhu, Fang Liu, Zhijie Nie, Yuchao Ye and Wenjin Yue

*

College of Biochemical Engineering; Anhui Polytechnic University; Wuhu - China

ABSTRACT

The aim of this work was to study the co-production of nattokinase and poly (γ-glutamic acid) by Bacillus subtilis

natto with soybean and rice husk under solid-state fermentation (SSF). The results showed that the size of soybean

particle and rice husk significantly improved the co-production of nattokinase and poly (γ-glutamic acid), yielding

2503.4 IU/gs and 320 mg/gs, respectively in the improved culture medium composed of 16.7% soybean flour and

13.3% rice husk with 70% water content. The yields increased by approximate 7- and 2-fold factor relative to their

original ones. Thus, the co-production of nattokinase and poly (γ-glutamic acid) under SSF could be considered as

an efficient method to exploit agro-residues for economical production of some higher-value products.

Key words: Co-production, Nattokinase, poly (γ-glutamic acid), Rice husk, Solid-state fermentation

*Author for correspondence: [email protected]

INTRODUCTION

Intravascular thrombosis, which results from blood

clot fibrin in arteries, is a bad disease against

human health. As compared to the clinical

thrombolytic drugs (urokinase and streptokinase),

nattokinase (NK) with many advantages such as

confirmed efficacy, prolonged effects, cost

effectiveness and preventative use is a potent

thrombolytic enzyme in vitro and in vivo (Zheng et

al. 2005). For example, it has four times

fibrinolytic activity over plasmin (Sumi et al.

1992), and the activity can be retained in the blood

for more than 3 h (Fujita et al. 1995). In view of

the traits like safe, low cost, and easy oral

administration, NK has been considered as an

excellent functional food for thrombosis therapy.

Poly (γ-glutamic acid) or γ-PGA is a

homopolymer of D- and L-glutamic acid units,

which is biodegradable, edible and nontoxic

toward humans and environment (Shih and Van

2001). Accordingly, it is a promising

environmental-friendly biomaterial with broad

industrial application in the fields of medicine,

foods, plastics, farming and many others (Shih and

Van 2001; Wang et al. 2008; Zhu et al. 2010).

Bacillus subtilis has been usually applied to

produce NK under submerged fermentation (SmF)

(Deepak et al. 2008) and to manufacture γ-PGA

under solid-state fermentation (SSF) (Wang, et al.

2008; Yao et al. 2012; Zeng et al. 2013). This

strain has been applied to co-produce multiple

biomaterials containing γ-PGA under SSF (Wang

et al. 2008; Yao et al. 2012; Zeng et al. 2013).

There are a large number of agricultural and

forestry residues produced world-wide whose

disposal is a serious problem in China. However,

these could be effectively used in SSF for enzyme

production (Jana et al. 2012; Selwal and Selwal

2012; Beniwal et al. 2013; Wang et al. 2013).

Co-producing NK and PGA


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Soybean and rice husk are low-cost and abundant

in China. Rice husk, in particular, is an excellent

solid support for SSF in view of its uniform shape

of the particles with fluffy and elastic

characteristics. On the other hand, γ-PGA

fermentation broth under SmF is highly viscous

and exhibits non-Newtonian rheology along with a

serious barrier against mixing, heat transfer and

oxygen supply (Richard and Margaritis 2003).

Impurities in the liquid fermentation increase the

cost for γ-PGA purification. As compared to SmF,

SSF can not only exploit cheap and easily

available raw agro-residues but also offers

economical and engineering advantages such as

low processing cost, less energy expenditure,

simple equipment and facilities (Pandey 2003;

Chen et al. 2005). γ–PGA, an important

composition of natto, can promote mineral

substance to be utilized. It also can preserve the

moisture of solid culture medium and further

improve NK production under SSF. Accordingly,

it is considered that co-production of NK and γ–

PGA under SSF should be an interesting work. To

our knowledge, the co-production, especially with

soybean and rice husk as solid matrix, has not yet

been reported. In this work, soybean and rice husk

were utilized as solid substrate and support for

SSF to co-produce NK and γ–PGA using Bacillus

subtilus natto.

MATERIAL AND METHODS

Material

NK-producing strain was obtained from the

commercial Bacillus subtilis natto powder and

maintained in 4ºC. Casein was purchased from

Sinopharm Chemical Reagent Co., Ltd. (SCRC),

China. Thrombin, cattle fibrinogen and urase were

bought from the National Institute for the Control

of Pharmaceutical and Biological Products

(NICPBF). Rice husk and Chinese

northeast soybean were bought from the local

market.

CO-production of NK And γ-PGA Under SSF

A dosage of 500 μL Bacillus subtilus natto

solution was inoculated in 150 g boiled Chinese

soybeans in a plastic case (Φ 20 cm). After being

mixed evenly, the solid culture medium was

covered with three layers of gauzes, then the case

was sealed with foodstuff preservative film with

six holes (Φ 2 mm) and incubated at 37ºC for 24 h.

Afterwards, the whole contents were preserved at

4ºC for 24 h.

Extraction of Crude NK And Determination Its

Activity

Sixteen gram fermented matrix was added in 40

mL normal saline in 250 mL flask, mixed at 100

rpm for 1 h and centrifuged (10,000 rpm, 10 min).

The above extraction protocols were carried out

two times. The supernatant was collected and

defined as crude NK. NK activity was determined

using the method of Wei et al. (2007). Briefly, 2.0

mL 0.5% casein solution and 1.0 mL crude NK

samples were incubated at 30ºC for 5 min, then the

NK sample was mixed with the casein solution

uniformly and incubated at 30ºC for 10 min.

Afterwards, 3.0 mL 10% trichloroacetic acid was

added in the mixture and incubated for 10 min,

then filtered. A control was prepared by mixing

1.0 mL crude NK sample with 3.0 mL 10%

trichloroacetic acid uniformly and then 2.0 mL

0.5% casein solution was added in the mixture.

Other protocols were the same as the above

mentioned. Then 1.0 mL NK sample and 1.0 mL

the control were added in two tubes containing 5.0

mL 0.55 mol/L sodium carbonate and 1.0 mL

phenol, respectively and incubated at 30ºC for 15

min after mixing well. The absorbance of the

sample and the control was read by an ultraviolet

spectrophotometer (SP-754, Shanghai Spectrum

Instruments Co Ltd, China) at 680 nm, which were

defined as As and Ac, respectively. NK activity

was calculated based on tyrosine calibration curve

and its achievable model (NK activity = K*(As-

Ac)*N*6/10, where K was the slope reciprocal of

tyrosine calibration curve, N was the distilled

fold). The unit of NK activity was defined as the

NK dosage required for 1.0 μg tyrosine formation

in 1 min. All the experiments were carried out in

three sets.

Extraction of Crude γ-PGA And Determination

Its Yield

The extraction method in this work was modified

based on the method by Bajaj et al.(2008). Twenty

gram fermented substrate was added in 80 mL

normal saline in 250 mL Bunsen beaker and mixed

at 30ºC on a rotary shaker (200 rpm) for 1 h, then

the mixed solution was filtered by two-layer gaze.

The filtrate was centrifuged at 10000 rpm for 15

min. Clarified supernatants were collected for γ-

PGA purification.

Yue, W. et al.


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γ-PGA was purified by the method reported by

Goto and Kuniko(1992). Ten milliliters of

supernatant was poured in 40 mL methanol and

kept at 4°C for 12 h. Crude γ-PGA was collected

by centrifugation at 10000 rpm and 4°C for 30 min

and then it was dissolved in distilled water. Any

insoluble impurity was removed by centrifugation.

The aqueous γ-PGA solution was desalted by

dialysis (molecular weight cut off 3500) against 1

L distilled water for 12 h with water exchange

three times. γ-PGA yield was determined by UV

assay according to the reported method previously

(Zeng et al. 2012). All the experiments were

carried out in three sets.

RESULTS AND DISCUSSION

Time courses of NK and γ-PGA production

Studies on finding the optimal time for NK and γ-

PGA production showed the maximum NK yield,

4.21 U/g dry substrate (U/gds or U/g) at 24 h (Fig.

1A). Afterwards, the yield began to decrease. As

shown in Figure1B, the γ-PGA yield exhibited a

trend from ascent to descent with the increment of

incubation time and the maximum yield was 144

mg/g at 36 h. The optimal time of γ-PGA

production was shorter than the previous report,

72 h (Bajaj et al. 2008).

Figure 1 - Schedule for NK (A) and γ-PGA (B)

production.

Effect of particle size of Soybean on NK and γ-

PGA Production

The effect of the particle size of solid substrate

(whole bean, quarter bean, and bean flour) on co-

production of NK and γ-PGA showed that the

maximum yields of NK and γ-PGA were obtained

with bean flour as substrate. It was inferred that

NK and γ-PGA production could be enhanced by

the small size of substrate particles.

Figure 2 - Effect of substrate particles size on NK (A)

and γ-PGA (B) production. ―Whole‖ referred to whole soybean as sold substrate under

SSF. ―Quarter‖ referred to smaller particle size of soybean

residue, which was one quarter, the size of whole soybean.

―Powder‖ referred to soybean flour. Inoculum size and water

size were 4 and 70%, respectively. The other conditions were

same as described in the ―Material and methods‖ section.

Smaller size of solid substrate particles provides

larger surface area. The enlarged area is helpful for

mass transfer by enhancing the affinity of substrate

to microbial cells. Thus, the substrate containing

small size particles gave higher yields of NK and

γ-PGA by meeting the rapid requirement of

microbial metabolism for nutrients, particularly at

the latter stage of metabolite synthesis. However,

the small size effect could also inhibit the

metabolite synthesis by blocking air to flow

fluently. It was deduced that nutrient rather than

oxygen was even more necessary for the microbial

metabolism at the latter stage. Nevertheless, the

poor oxygen concentration available in solid

matrix has a harmful effect on microbial

metabolism. Therefore, it is important that a

desired balance between particle size and air-flow

rate is established in SSF processes.

Effect of Rice Husk on NK And γ-PGA

Production

The effect of particle size of rice husk on air-flow

was studied, the result indicated that rice husk can

improve the strain in co-producing NK and γ-

PGA. The improvement was more prominent for



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NK production than for γ-PGA production (Fig.

3). Figure 4 showed that the NK yield kept on

decreasing with the increment of rice husk content.

On the contrary, the yield of γ-PGA kept on

increasing until the ratio of rice husk to soybean

grew up to 5:4, when the maximum yield (227.6

mg/g) was achieved.

Figure 3 - NK (a) and γ-PGA (b) production using rice

husk. In this diagram, ―Addition of rice husk‖ refers to direct

addition 30 g rice husk in the solid fermentation medium

containing 150 g soybean flour, ―None‖ refers to the solid

fermentation medium without any rice husk. Inoculum size

and water size were 4 % and 70%, respectively. The other

conditions were seen in the ―Material and methods‖ section.

Figure 4 - Effect of the ratio of bean flour to rice husk

on NK and γ-PGA production. In this experiment, the solid fermentation medium was

composed of 30 g soybean flour and different amount of rice

husk decided by the ratio of bean flour to rice husk. Inoculum

size and water size were 4 % and 70%, respectively. NK

activity was assayed after the strain was incubated for 24 h,

and γ-PGA yield was determined after the strain was

incubated for 36 h. The other conditions were seen in the

―Material and methods‖ section.

Accordingly, it was concluded that the

enhancement in air flow could enhance the co-

production of NK and γ-PGA. In view of its fluffy

and elastic nature, rice husk could make the

compact culture medium (composed by fine

soybean particles) so fluffy that countless new

―air-lines‖ are can be constructed. Besides, rice

husk can enlarge the binding area of microbial cell

to substrate by making an even and extensive

distribution of microorganism in solid culture

medium. This not only improved the air

permeability and heat dissipation in the solid

medium but also speeded up mass transfer of

substrate and product. The fermented rice husk

could be utilized to improve organic fertilizer in

water retention and nutrients (Chen et al. 2005).

This was in agreement with the present increasing

emphasis on exploiting agro residues. Therefore,

rice husk could be regarded as an excellent

leavening agent for SSF.

Effects of Water Content and Inoculum Size on

NK andγ-PGA Production

In view of moisture retention of γ-PGA, different

water contents (50-90%) were investigated to find

the optimum of moisture level. Figure 5 indicated

that the water content at 70% was the best initial

moisture level for NK and γ-PGA production.

Figure 5 - Effect of the water content on NK and γ-

PGA production. In this experiment, the solid fermentation medium was

composed of 30 g soybean flour and 18 g rice husk. Inoculum

size was determined as 4%. NK activity was assayed after the

strain was incubated for 24 h, and γ-PGA yield was

determined after the strain was incubated for 36 h. The other


Yue, W. et al.


722

Water content in solid culture medium has a direct

correlation with the water activity, which is a

critical factor for microbial growth and

metabolism under SSF. Poor water can limit some

nutrients to be dissolved and transferred so as to

inhibit microbial growth, while excessive water

can inhibit the microbial growth by making

substrate clustered and preventing air permeability

and heat dissipation (Yadav et al. 2008). It’s worth

noting that γ-PGA has a capability of water

retention to reduce the evaporation of the moist

culture medium during incubation. On the other

hand, water in the fluffy culture medium

composed of rice husk could evaporate faster than

that in the compact medium. Therefore, the

optimization of water content in the culture

medium is essential. Generally, the inoculum size

has a dramatical effect on microbial biomass and

metabolites synthesis. The effect of inoculum size

(2-12%) was explored on the co-production of NK

and γ-PGA. Figure 6 indicated that the maximum

yields of NK and γ-PGA were achieved with the

inoculum sizes of 4 and 8%, respectively.

Excessive inoculum size could inhibit NK

synthesis, but less inoculum size would not

stimulate γ-PGA production completely.

Accordingly, the inoculum size at 6% was chosen

in further studies.

Figure 6 - Effect of inoculum size on NK and γ-PGA

production. In this experiment, the solid fermentation medium was

composed of 30 g soybean flour and 18 g rice husk. Water

content was determined as 70%. NK activity was assayed

after the strain was incubated for 24 h, and γ-PGA yield was

determined after the strain was incubated for 36 h. The other


Table 1 - Analysis of the difference of improved NK and γ-PGA production from the original ones along with

reaction time.

Reaction

time (h)

Improved Original

RNK RPGA NKs

(U/g)

γ-PGAs

(mg/g)

NKp (%) γ-PGAp

(%)

R Top NKs

(U/g)

Topγ-PGAs

(mg/g)

9 1.67 65.6 52.22 45.40 1.15

0.40 0.45

18 3.14 126.2 98.58 87.34 1.13 0.75 0.87

24 6.69 147.25 209.70 101.90 2.06 4.21 1.59 1.02

27 6.08 177.8 190.49 123.04 1.55 1.44 1.23

36 3.70 222.6 116.13 154.05 0.75 144.5 0.88 1.54

45 3.57 194.8 111.95 134.81 0.83 0.85 1.35

54 1.99 153 62.24 105.88 0.59 0.47 1.06

63 1.13 127.6 35.51 88.30 0.40 0.27 0.88

72 0.77 85.2 24.23 58.96 0.41 0.18 0.59

Mean 3.19 144.45

NKs and γ-PGAs denote the yields of NK and γ-PGA, respectively. NKp and γ-PGAp are their percents relative to their

corresponding means, respectively. R equals to the ratio of NKp to γ-PGAp. Top NKs and Top γ-PGAs are the top yields of NK

(incubated for 24 h) and γ-PGA (incubated for 36 h) before improvement, respectively. RNK and RPGA are the ratio of NKs to top

NKs and the ratio of γ-PGAs to top γ-PGAs, respectively. In the improved experiment, the solid fermentation medium was

composed of 30 g soybean flour and 18 g rice husk. Inoculum size and water content were designed as 6% and 70%, respectively.

The other conditions were seen in the ―Material and methods‖ section.

Inoculum size is very crucial for metabolite

synthesis under SSF. Poor inoculum size can defer

the synthetic progress of metabolite by prolonging

the time course of microbial growth, while

superfluous inoculum size can give rise to major

nutrients of culture medium to be exhausted in

microbial growth (Kashyap et al. 2002). Sufficient

inoculation is beneficial to both the cell growth

and product synthesis (Zeng et al. 2013).

Accordingly, it is important for achieving a

promising yield of metabolite to keep the balance

between the biomass proliferating and available

substrate(Pandey et al. 2000; Kashyap, et al.

2002). Rice husk and the small size effect of



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soybean have a positive effect on microbial

distribution, and this reasonably accelerates the

growth of microbial cell in this work. It was

concluded that the inoculum size depended on not

only the dispersability of culture medium but also

microbial distribution.

B. subtilus natto was incubated in an optimized

culture medium for 9-72 h based on the above

results. It was found that NK and γ-PGA yields at

24-45 h were higher than their corresponding

values at 9-72 h. NK and γ-PGA yields at 24 and

27 h surpassed the maximum ones without

optimization. The ratio of NKp to γ-PGAp (R) at 27

h was closer to one relative to that at 24 h (Table

1). Consequently, the optimum incubation time

was considered as 27h for the co-production of

NK and γ-PGA, where NK and γ-PGA yields were

6.08 U/g and 177.8 mg/g, respectively, and the

corresponding fibrinolytic activity of NK was

1564.4 IU/g (assayed by the standard fibrin plate

method(Astrup and Müllertz 1952)). The

fibrinolytic activity of NK and γ-PGA yield was

converted to 2503.4 IU/g for soybean (IU/gs) and

320 mg/gs, increasing by approximate 7 and 2-fold

factors relative to their original yields (342.98

IU/gs and 144.5 mg/gs), respectively, when

soybean instead of total solid supports was

regarded as a measurement baseline. Furthermore,

the γ-PGA yield in this work was higher than that

(51.3 mg/g) in co-production of iturin A and γ-

PGA (Yao et al. 2012). Also, the NK and γ-PGA

yields were higher than the ones reported by Zeng

et al.(2013). Troublesomely, it is difficult to

extract NK from the co-production ferment

including γ-PGA because high concentration of γ-

PGA could disturb NK separation (Wei et al.

2012). Microbial γ-PGA with relatively high

molecular weight can make the ferments too

viscous to be separated easily. Therefore, it is very

necessary to construct an efficient method for the

departure of NK from γ-PGA in the further work.

CONCLUSIONS

In summary, the smaller size of the substrate

particles significantly improved the co-production

of NK and γ-PGA by B. subtilus natto. Rice husk

enhanced the soybean utilization and decreased the

detrimental effect of the smaller size of the

substrate particles related to aeration. The

fermented solid medium could be utilized to

prepare organic fertilizer. These findings

suggested that the co-production of NK and γ-

PGA under SSF with agro-residues as substrate

could be considered as an efficient method to

exploit agro-residues for economical production of

higher-value products.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial

support granted to this work by National Natural

Science Foundation of China (51202002), Key

Project of Provincial Natural Science Research of

Colleges and Universities in Anhui Province

(KJ2014A025), and National Training Programs

of Innovation and Entrepreneurship for

Undergraduates (201210363042, 201310363076).

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Received: April 02, 2015;

Accepted: May 25, 2015.

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